This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: An important application of cryo-electron tomography is to identify and determine the orientation and conformation of large macromolecular assemblies in their native cellular environment. This requires the development of algorithms that can efficiently detect target structural motifs within noisy tomographic 3-D density maps, using probe structures provided by single-particle cryo-EM or x-ray crystallography. Dr. Renken completed his project to improve the implementation of motif searching within SPIDER using RAMOS (Rapid Motif Search: Rath et al., 2004, J. Struct. Biol. 145, 84-90). RAMOS is an algorithm that uses local variance in order to calculate the locally normalized cross-correlation function between a small motif and a larger volume. The new project entailed determination of the location and orientation of ryanodine receptors (RyR) on vesicles of sarcoplasmic reticulum (SR) within tomographic reconstructions of frozen-hydrated triad junctions from skeletal muscle. The result was a partial map of the calcium release unit, showing new details such as the paracrystalline nature of calsequestrin (CSQ) in the vicinity of the RyR, and a dome-shaped vestibule between the RyR pore and the SCQ layer. The results were published, with a cover picture, in a paper highlighted in last year's annual progress report (Renken et al. 2008. Structure of Frozen-hydrated Triad Junctions: A Case Study in Motif Searching Inside Tomograms. J. Struct. Biol. 165: 53-63. PMCID 2655133).